The cell cycle in eukaryotic organisms is a highly regulated network of processes that contains numerous checks and balances to ensure its proper and timely execution. Several mechanisms, called checkpoints, exist to ensure that the events during a specific period of the cell cycle are carried out appropriately before proceeding to the next phase of the cell cycle. The checkpoint mechanisms of interest in this proposal are those that respond to DNA damage and to problems arising during DNA replication. These checkpoints function to temporarily halt or slow down the cell cycle in order to correct problems with genome integrity. Defects in the checkpoint mechanisms result in increased chromosome instability and such defects are known cancer predisposition conditions in human. These pathways have been conserved from yeast to human which makes the former an ideal organisms for basic checkpoint studies. Two classes of protein kinases are critical for signal transduction. The yeast Mec1 kinase is related to the ATR and ATM kinases, the latter being defective in ataxia telangiectasia patients; it functions most proximal to sites of DNA damage and to stalled replication forks. The Rad53 protein kinase, related to human Chk1 and Chk2, acts downstream in halting the cell cycle and activating other response pathways. The catalytic centers of these protein kinases are activated during checkpoint progression. While numerous genetic and molecular biological studies have identified many of the factors involved in the various checkpoints, the biochemical mechanisms of activation of Mec1 and of Rad53 have remained relatively obscure. The biochemical studies in this proposal are aimed at understanding Mec1 activation by the replication initiation protein Dpb11, called TopBP1 in human and Rad4/Cut5 in fission yeast (Aim 1), to study the nature of activation of Mec1 by structure-function analysis (Aim 2), and to study the role of DNA binding in activation of Rad53 (Aim 3). These biochemical experiments will be complemented by genetic studies to obtain an integrated view of checkpoint activation. PUBLIC HEALTH RELEVANCE In human cells, so-called checkpoints monitor the intactness of the DNA, our genetic material, and the correctness of DNA duplication prior to cell division. Patients with known defects in checkpoint pathways are at a highly increased risk for developing cancer. Checkpoint pathways are conserved from human to yeast, and we are proposing to study these pathways in yeast, because as a model organism it is more approachable to genetic and biochemical analysis.